Summary/Abstract In transformed cells the demand to accumulate biomass requires substantial metabolic pathway alteration. Understanding this altered metabolism will enable identification of liabilities that can be exploited for cancer therapy. In prior work, we found that hyperoxic stress is a considerable force driving metabolic pathway dependence in breast cancer. Indeed, the enzyme most differentially required in high versus low oxygen environments, NFS1, is required for breast cancer metastasis to the lung. Moreover, NFS1 lies in an amplified region under positive selection in lung adenocarcinoma. Therefore, from prior work we conclude that the high oxygen environment of the lung is a key metabolic factor to which incipient breast metastases and lung tumors uniquely adapt, in part, via NFS1. Ours is the first work to describe a role for this critical pathway in cancer. The purpose of this grant is to gain mechanistic understanding of the NFS1 requirement, focusing on basal- like breast cancer (BLBC). NFS1 is a key enzyme in the biosynthesis of iron-sulfur clusters (ISC), essential cofactors in 48 proteins in humans. We found that BLBC cell lines are strikingly more sensitive than luminal lines to suppression of NFS1. We propose to identify the mechanistic underpinnings of this observation, and extend our findings to other cancer subtypes. We will suppress ISC containing proteins in a panel of breast cancer cell lines and verify which are differentially required in BLBC. Validated targets will be inhibited in xenograft-based models of breast cancer and metastasis to assess their impact on these processes. Many ISC containing genes are involved in the maintenance of genomic integrity. Our preliminary work reveals that NFS1 suppression results in the formation of double strand DNA breaks. These observations led us to suppress POLE, a key genomic integrity enzyme. Interestingly, suppression of POLE also blocks proliferation and induces double strand breaks in BLBC cell lines far more than luminal lines. Prior work has indicated that BLBC has defects in aspects of DNA repair that may sensitize them to therapy. Therefore we will suppress POLE and assess the impact on DNA replication, origin firing, replication fork stalling and restarting, and the repair of damaged replication forks. These experiments will contribute to our fundamental understanding of the sensitivity of BLBC to inhibition of DNA replication and induction of DNA damage. Finally, NFS1 suppression sensitizes cells to oxidative damage via a non-apoptotic form of cell death termed ferroptosis. Our findings lead to the intriguing possibility that activation of the iron-starvation response in iron-replete conditions, thereby tricking cancer cells into taking up excess iron, will render them highly susceptible to further oxidative stress and death by ferroptosis. By mechanistically understanding how best to activate the iron-starvation response downstream of NFS1, we anticipate that we will gain the benefit of inducing increased sensitivity to oxidants without having to target a generally cell-essential pathway.